The endothelium is the monolayer of cells, located at the posterior of the cornea, which maintains corneal transparency. In humans, corneal endothelial cell density decreases with age, suggesting that the rate of cell replacement by mitosis does not keep pace with the rate of cell loss. Other factors, such as diabetes, inflammation, ocular trauma or surgery, contribute to cell loss and can lead to endothelial decompensation, stromal edema, and loss of visual acuity. Bullous keratopathy caused by endothelial dysfunction is currently medically untreatable and restoration of vision can only be accomplished by corneal transplantation. Our long- term goal is to develop medical treatments to promote healing of stressed corneal endothelium. To reach this goal, we must discover how corneal endothelial repair is regulated. Normally, both mitosis and cell movement contribute to monolayer repair; however, human corneal endothelium does not readily divide upon injury, so repair occurs mainly by cell movement. This relative lack of mitotic capability is not universal, since corneal endothelial cells in species, such as rabbits, readily divide. Studies during the last grant cycle focussed on the regulation of cell movements. The proposed studies will change focus to concentrate on the regulation of the corneal endothelial cell cycle. This approach is more direct and may be more helpful in achieving the long-term goal. The working hypothesis for these studies is that, although in humans the relative number of senescent, non-dividing cells increases with age, there is, at all ages, a population of mitotically quiescent, G1-phase arrested cells which are capable of mitogenic stimulation. In both humans and rabbits, in vivo conditions maintain the endothelium in a quiescent, differentiated state to preserve its important physiologic functions. Factors which may contribute to quiescence include contact inhibition, the relatively high concentration of TGF-beta (a potential growth inhibitor) in the aqueous humor, and autocrine mitotic inhibition by PGE2. Reversal of growth arrest in the presence of growth factors, such as EGF, should promote mitosis in stressed corneal endothelium. The proposed studies will use cell biological, pharmacological and molecular biological methods to achieve the following Specific Aims: 1) Compare in human and rabbit corneal endothelium the relative percent of actively cycling and quiescent cells and the relative position within the cell cycle in which quiescent cells are arrested, 2) Determine whether, during early fetal development, there is a change in human corneal endothelium from an actively cycling to a quiescent state which correlates with the formation of a stable, contact inhibited and/or differentiated monolayer, 3) Determine whether release from contact inhibition, particularly in the presence of EGF and/or indomethacin can stimulate corneal endothelial cells to re-enter the cell cycle, and 4) Determine whether TGF-beta inhibits corneal endothelial cell division and whether such inhibition is reversible. These studies should help determine the relative mitotic potential of human corneal endothelium, investigate causes for in vivo inhibition of corneal endothelial mitosis and help discover means to reverse mitotic inhibition in a clinically relevant manner.